Our long term goal is to understand how effector proteins are involved in normal cellular processes and in mechanisms of pathophysiology. The aim of this proposal is to examine the characteristics and mechanism of action of a set of relatively tissue-specific calcium modulated proteins (S100) that may function as intracellular effector proteins. Only some of the S100 proteins have been isolated and characterized, and the relative amounts of each S100 protein in a given species or tissue have not been determined. The physiological functions of S100 proteins are not known and there are specific assays for individual S100 components. The proposed studies are a purification and characterization of rat S100 proteins and analyses of the levels, subcellular distribution, regulation and mechanism of action of these proteins using quantitative cell biological and biochemical approaches. Emphasis is on the development and refinement of methods for study of S100 Beta to allow unequivocal interpretation of biological studies. The biological systems are rat brain and the C6 cell line derived from a rat glial tumor induced by the carcinogen, N-nitrosomethylurea. We will purify S100 proteins to homogeneity from rat brain, determine their biochemical and immunochemical properties, and compare them to the analogous proteins purified from C6 cells. We will develop specific immunochemical assays for each S100 protein by various approaches. These approaches have resulted in the recent production of antibodies specific for the S100 Beta polypeptide of brain S100 fractions. Using biochemical cytology methods, we will determine the levels and subcellular distribution of S100 proteins in C6 cells under various physiological states and culture conditions. By a direct mechanistic approach, we have detected molecular targets of S100 Beta. We will further investigate these targets, including subcellular distribution and possible biochemical activities. These studies might yield insight into common and unique mechanisms of regulation by calcium modulated proteins and will provide the necessary methods and rigorous foundation for future studies on molecular mechanisms of action of these proteins and for current studies of human disease states.